Compositions and methods for detection of hepatitis a virus nucleic acid

ABSTRACT

Nucleic acid oligomeric sequences and in vitro nucleic acid amplification and detection methods for detecting the presence of HAV RNA sequences in samples are disclosed. Kits comprising nucleic acid oligomers for amplifying and detecting HAV nucleic acid sequences are disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/031,353, filed Sep. 19, 2013, which is a continuation of U.S. patentapplication Ser. No. 13/243,243, filed Sep. 23, 2011, now U.S. Pat. No.8,563,707, which is a continuation of U.S. patent application Ser. No.12/429,589, filed Apr. 24, 2009, now U.S. Pat. No. 8,063,197, which is adivisional of U.S. patent application Ser. No. 11/182,177, filed Jul.13, 2005, now U.S. Pat. No. 7,544,792, and claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Patent Application No. 60/587,734,filed Jul. 13, 2004, all of which are incorporated herein by referencein their entirety.

FIELD

This invention relates to diagnostic detection of a human virus, andspecifically relates to assays to detect human hepatitis A virussequences by using in vitro nucleic acid amplification and detection ofamplified sequences.

BACKGROUND

Hepatitis A virus (HAV) is the causative agent of one form of hepatitisthat may produce symptoms that include fever, fatigue, nausea, abdominalpain, diarrhea, loss of appetite, and jaundice over less than twomonths. Of those infected with HAV, about 10% to 15% have a prolonged orrelapsing symptoms over a six to nine months following infection.Immunity to HAV, based on the individual's production of anti-HAVimmunoglobulin G (IgG), follows both symptomatic and asymptomaticinfections.

Although the incidence of HAV infections has dramatically decreased inparts of the world in which vaccination for HAV (e.g., by usinginactivated HAV) has been widely used since the late 1990's, epidemicsof HAV infections (greater than 700 cases per 100,000 population) mayoccur in non-immune populations where poor sanitary conditions exist,even temporarily, e.g. following an earthquake. HAV is shed in feces ofinfected persons and is usually transmitted by the fecal-oral route.Community-wide outbreaks may result from food-borne transmission thatoccurs when an HAV-infected food handler contaminates food duringpreparation, or when food materials are contaminated during growing,harvesting, packing, or processing in the distribution system.Transmission may also result from contact with HAV-contaminated serum,blood products, or contaminated needles, e.g., by transfusion orinjection drug use. Persons at risk of HAV infection include those whohave household or sex contacts with HAV-infected persons, persons whohave clotting-factor disorders (e.g., hemophilia) or chronic liverdisease, persons who travel in countries here hepatitis A is common, menwho have sex with men, illegal drug users, and children who live inareas with high rates of hepatitis A (e.g., >20 cases per 100,000population).

HAV is a 27-nm RNA virus (picornavirus) that contains a plus-sensesingle-stranded RNA genome of about 7.5 kb, for which a single serotypehas been found worldwide. HAV replicates in the liver, is excreted inbile, and is shed in feces during the acute phase of an infection (up to10.sup.8 virus per ml). The incubation period is usually two to sixweeks before symptoms appear. Diagnosis of hepatitis A cannot bedifferentiated from other types of viral hepatitis by symptoms or otherclinical features (e.g., elevated serum aminotransferase levels).Typically, hepatitis A diagnosis is confirmed by serological testingthat provides positive results for the presence of anti-HAVimmunoglobulins (Ig). Anti-HAV IgM is generally present five to ten daysbefore the onset of symptoms and is undetectable in most patients by sixmonths later, whereas anti-HAV IgG appears early during infection andremains detectable for the individual's lifetime. HAV RNA can bedetected in the blood and stool of most persons during the acute phaseof infection by using nucleic acid testing methods, e.g., amplificationby the polymerase chain reaction (PCR), and nucleic acid sequencing,which has been used to identify the genetic relatedness of HAV followingcommunity-wide infections (Dato et al., Morbidity Mortality Wkly. Rpt.,2003, 52(47): 1155-57; LaPorte et al., Morbidity Mortality Wkly. Rpt.,2003, 52(24): 565-67). These methods, however, are not generally usedfor diagnostic purposes.

In the USA, about 100 persons die each year from acute liver failure dueto hepatitis A (death rate of about 0.015%). Even in nonfatal hepatitisA cases there are substantial costs associated with HAV infections,including the costs of patient hospitalization, outpatient visits, andlost workdays. Public health costs associated with hepatitis A outbreaksinclude locating and administering immune globulin to people exposed toan infected individual or infectious source (e.g., contaminated water orfood) within two weeks of exposure. Substantial psychological costs andeconomic losses may result from the perceived risk of infection,particularly for community-wide outbreaks. Because of the relative easeof HAV transmission in contaminated food and water, and the morbidityassociated with hepatitis A, HAV is a potential agent for use inbiological terrorism.

There exists a need to accurately detect the presence of HAV inbiological and environmental samples. There exists a need to rapidlydiagnose HAV-infected individuals. For example, because immune globulinmust be administered to a person within two weeks of HAV exposure to beeffective, there exists a need for a rapid and accurate assay topromptly evaluate food handlers with hepatitis symptoms and reportHAV-positive sources to public health agencies. There is a need todetect HAV present in contaminated materials, such as water and food, toprevent community-wide outbreaks or epidemics resulting from use orconsumption of these materials. There is also a need to detect HAVcontamination in products that may be used in medical treatment, e.g.,blood or serum used for transfusions or for the manufacture of factorsderived from human fluids.

The present invention responds to these needs by disclosingoligonucleotide sequences used in nucleic acid testing methods to detectthe presence of HAV nucleic acid (HAV RNA or cDNA derived from RNA) in asample.

SUMMARY

The invention includes nucleic acid oligomers useful for purification,amplification and detection of HAV target sequences. Such oligomers orcombinations of oligomers may be contained in a kit configuration,embodiments of which may include additional oligomers and/or otherreagents for amplifying and/or detecting a HAV sequence. The inventionalso includes methods of detecting HAV in a sample that use steps ofpurifying HAV nucleic acid from other components in the sample,amplifying a HAV RNA target sequence or cDNA made therefrom by using anucleic acid polymerase in vitro and any combination of amplificationoligomers as described herein to produce an amplified product, anddetecting the amplified product by using a detection probe thathybridizes specifically with at least a portion of the amplifiedproduct. In one embodiment, HAV nucleic acid is purified by using atleast one capture oligomer that includes a sequence that hybridizesspecifically to a HAV RNA target region to form a hybridization complexthat includes the HAV RNA being separated from other sample components.

One aspect of the invention is a combination of at least two oligomersspecific for amplifying a HAV target region that include: for a firstHAV target region, an oligomer of about 23 to 26 nt contained in thesequence of SEQ ID NO:138 that includes at least the sequence of SEQ IDNO:139 or SEQ ID NO:140, or an oligomer in a size range of about 19 to25 nt contained in the sequence of SEQ ID NO: 141 and that contains atleast one sequence of SEQ ID NOS:142 to 146, or a promoter primeroligomer in a size range of about 50 to 53 nt that includes a HAVtarget-specific portion of any one of SEQ ID NOS:21 to 27; for a secondHAV target region, an oligomer of about 21 to 27 nt contained in thesequence of SEQ ID NO:60 or contained in the sequence of SEQ ID NO:86that includes at least the sequence of SEQ ID NO:156, or a promoterprimer oligomer in a size range of about 48 to 54 nt that includes a HAVtarget-specific portion of any one of SEQ ID NOS:29 to 32; for a thirdHAV target region, an oligomer of about 24 to 30 nt contained in thesequence of SEQ ID NO:147 that includes at least the sequence of SEQ IDNO:148, or is contained in the sequence of SEQ ID NO:157 and thatincludes at least the sequence of SEQ ID NO:158, or a promoter primeroligomer that includes a HAV target-specific portion of SEQ ID NO:31 orSEQ ID NO:32; for a fourth HAV target region, an oligomer of about 18 to27 nt contained in the sequence of SEQ ID NO:93 or SEQ ID NO:95 and thatcontains at least the sequence of SEQ ID NO:97, SEQ ID NO:159, or SEQ IDNO:160, or a promoter primer oligomer that includes a HAVtarget-specific portion of SEQ ID NO:33; for a fifth HAV target region,an oligomer of about 19 to 31 nt contained in the sequence of SEQ IDNO:149 and that includes at least the sequence of SEQ ID NO:150, or apromoter primer oligomers in a size range of about 51 to 56 nt thatincludes a HAV target-specific portion of any one of SEQ ID NOS:34 to40; for a sixth HAV target region, an oligomer of about 24 to 28 ntcontained in the sequence of SEQ ID NO:161 and that include at least thesequence of SEQ ID NO:162, or a promoter primer oligomer that includes aHAV target-specific portion of SEQ ID NO:41 or SEQ ID NO:42; for aseventh HAV target region, an oligomer of about 20 to 30 nt contained inthe sequence of SEQ ID NO: 151 and that includes at least any one of thesequences of SEQ ID NO:152 to SEQ ID NO:155, or is contained in SEQ IDNO:163 and includes at least the sequence of SEQ ID NO:164, or iscontained in SEQ ID NO:165 and includes at least any one of thesequences of SEQ ID NOS:166 to 168, or a promoter primer oligomer in asize range of about 51 to 56 nt and that includes a HAV target-specificportion of any one of SEQ ID NOS:43 to 49; and a second amplificationoligomer. Preferred embodiments of combinations of at least twooligomers specific for the first HAV target region are selected from SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ IDNO:85, SEQ ID NO:143, SEQ ID NO:144, and SEQ ID NO:145. Preferredembodiments of combinations of at least two oligomers specific for thesecond HAV target region are selected from SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:58, SEQ ID NO:59,SEQ ID NO:60, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ IDNO:156. Preferred embodiments of combinations of at least two oligomersspecific for the third HAV target region are selected from SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:89, SEQ ID NO:90,SEQ ID NO:91, and SEQ ID NO:148. Preferred embodiments of combinationsof at least two oligomers specific for the fourth HAV target region areselected from SEQ ID NO:33, SEQ ID NO:63, SEQ ID NO:92, SEQ ID NO:93,SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, and SEQ ID NO:97. Preferredembodiments of combinations of at least two oligomers specific for thefifth HAV target region are selected from SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68,SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:97, SEQ ID NO:149, and SEQ IDNO:150. Preferred combinations of at least two oligomers specific forthe sixth HAV target region are selected from SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:98, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:161, and SEQ ID NO:162. Preferred combinations of atleast two oligomers specific for the seventh HAV target region areselected from SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:73, SEQ ID NO:74,SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79,SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:152, SEQ ID NO:153, SEQID NO:155, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166,SEQ ID NO:167, and SEQ ID NO:168.

Other preferred embodiments further comprising at least one captureprobe oligomer selected from the group consisting of SEQ ID NOS:1 to 7and SEQ ID NOS:8-14 linked to a moiety that allows it to bind a solidsupport. In one aspect of this embodiment, the binding moiety is asubstantially homopolymeric nucleic acid sequence. In one aspect of thisembodiment, the solid support is a magnetic bead. Still otherembodiments further include at least one detection probe oligomerselected from SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ IDNO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121 to SEQ ID NO:124,and SEQ ID NO:126 to SEQ ID NO:130. Preferred embodiments ofcombinations of oligomers include at least two oligomers specific foramplifying a selected HAV target region and at least one detection probeoligomer that is specific for a sequence contained in HAV genomicsequence located between the selected two oligomers specific foramplifying the selected HAV target region. Preferred embodiments of suchcombinations of oligomers may be packaged together in a kit, which mayfurther contain other reagents such as reagents used in purifying HAVRNA from a sample and/or reagents used in in vitro nucleic acidamplification, and/or reagents used in producing a detectable signalfrom a detection probe oligomer.

Another aspect of the invention is a method of detecting the presence ofHAV in a sample that includes the steps of purifying HAV nucleic acidfrom other components in a sample containing HAV; amplifying a HAVtarget sequence in the purified HAV nucleic acid, or a cDNA madetherefrom, by using an in vitro amplification reaction that includes atleast two amplification oligomers specific for a selected HAV targetregion, which include: for a first HAV target region, an oligomer ofabout 23 to 26 nt contained in the sequence of SEQ ID NO:138 and thatincludes at least the sequence of SEQ ID NO:139 or SEQ ID NO:140, or anoligomer in a size range of about 19 to 25 nt contained in the sequenceof SEQ ID NO: 141 and that contains at least one sequence of SEQ IDNOS:142 to 146, or a promoter primer oligomer in a size range of about50 to 53 nt that includes a HAV target-specific portion of any one ofSEQ ID NOS:21 to 27; for a second HAV target region, an oligomer ofabout 21 to 27 nt contained in the sequence of SEQ ID NO:60 or containedin the sequence of SEQ ID NO:86 and that includes at least the sequenceof SEQ ID NO:156, or a promoter primer oligomer in a size range of about48 to 54 nt that includes a HAV target-specific portion of any one ofSEQ ID NOS:29 to 32; for a third HAV target region, an oligomer of about24 to 30 nt contained in the sequence of SEQ ID NO:147 and that includesat least the sequence of SEQ ID NO:148, or contained in the sequence ofSEQ ID NO:157 and that includes at least the sequence of SEQ ID NO:158,or a promoter primer oligomer that includes a HAV target-specificportion of SEQ ID NO:31 or SEQ ID NO:32; for a fourth HAV target region,an oligomer of about 18 to 27 nt contained in the sequence of SEQ IDNO:93 or SEQ ID NO:95 and that contains at least the sequence of SEQ IDNO:97, SEQ ID NO:159, or SEQ ID NO:160, or a promoter primer oligomerthat includes a HAV target-specific portion of SEQ ID NO:33; for a fifthHAV target region, an oligomer of about 19 to 31 nt contained in thesequence of SEQ ID NO:149 and that includes at least the sequence of SEQID NO:150, or a promoter primer oligomer in a size range of about 51 to56 nt that includes a HAV target-specific portion of any one of SEQ IDNOS:34 to 40; for a sixth HAV target region, an oligomer of about 24 to28 nt contained in the sequence of SEQ ID NO:161 and that includes atleast the sequence of SEQ ID NO:162, or a promoter primer oligomer thatincludes a HAV target-specific portion of SEQ ID NO:41 or SEQ ID NO:42;for a seventh HAV target region, an oligomer of about 20 to 30 ntcontained in the sequence of SEQ ID NO: 151 and that includes at leastany one of the sequences of SEQ ID NO:152 to SEQ ID NO:155, or iscontained in SEQ ID NO:163 and includes at least the sequence of SEQ IDNO:164, or is contained in SEQ ID NO:165 and includes at least any oneof the sequences of SEQ ID NOS:166 to 168, or a promoter primer oligomerin a size range of about 51 to 56 nt and that includes a HAVtarget-specific portion of any one of SEQ ID NOS:43 to 49; and a secondamplification oligomer, to produce an amplified product of the selectedHAV target region; and detecting the amplified product. Preferably, thedetecting step is performed by using a nucleic acid detection probecomprising a target hybridizing region that hybridizes specifically withat least a portion of the amplified product.

A preferred embodiment in the purifying step contacts the sample with atleast one capture probe oligomer selected from the group consisting ofSEQ ID NOS:1 to 7 and SEQ ID NOS:8-14 linked to a moiety that allows itto bind a solid support, wherein a target hybridization sequence of saidat least one capture probe oligomer hybridizes specifically to asequence in HAV RNA to form a hybridization complex with the HAV RNA,and can be used with a solid support to separate the HAV RNA from othersample components. Preferred embodiments that amplify a sequence in thefirst HAV target region use at least two oligomers specific for thefirst HAV target region selected from SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:80, SEQ ID NO:81, SEQ IDNO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:143, SEQ IDNO:144, and SEQ ID NO:145; and then use at least one detection probethat hybridizes specifically to the amplified product of the first HAVtarget region. Preferred embodiments that amplify a sequence in thesecond HAV target region use at least two oligomers specific for thesecond HAV target region selected from SEQ ID NO:28, SEQ ID NO:29, SEQID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:156; andthen use at least one detection probe that hybridizes specifically tothe amplified product of the second HAV target region. Preferredembodiments that amplify a sequence in the third HAV target region useat least two oligomers specific for the third HAV target region selectedfrom SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:89, SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:148; and then use atleast one detection probe that hybridizes specifically to the amplifiedproduct of the third HAV target region. Preferred embodiments thatamplify a sequence in the fourth HAV target region use at least twooligomers specific for the fourth HAV target region selected from SEQ IDNO:33, SEQ ID NO:63, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ IDNO:95, SEQ ID NO:96, and SEQ ID NO:97; and then use at least onedetection probe that hybridizes specifically to the amplified product ofthe fourth HAV target region. Preferred embodiments that amplify asequence in the fifth HAV target region use at least two oligomersspecific for the fifth HAV target region selected from SEQ ID NO:34, SEQID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:97, SEQ ID NO:149, and SEQID NO:150; and then use at least one detection probe that hybridizesspecifically to the amplified product of the fifth HAV target region.Preferred embodiments that amplify a sequence in the sixth HAV targetregion use at least two oligomers specific for the sixth HAV targetregion selected from SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:161, and SEQID NO:162; and then used at least one detection probe that hybridizesspecifically to the amplified product of the sixth HAV target region.Preferred embodiments that amplify a sequence in the seventh HAV targetregion use at least two oligomers specific for the seventh HAV targetregion selected from SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:104, SEQID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:152, SEQ ID NO:153,SEQ ID NO:155, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ IDNO:166, SEQ ID NO:167, and SEQ ID NO:168; and then use at least onedetection probe that hybridizes specifically to the amplified product ofthe seventh HAV target region. In one aspect of this preferred method,the nucleic acid detection oligomer comprises a target hybridizingregion nucleic acid sequence consisting essentially of a nucleic acidsequence selected from the group consisting of SEQ ID NO:109, SEQ IDNO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQID NO:121 to SEQ ID NO:124, and SEQ ID NO:126 to SEQ ID NO:130. In oneaspect of this preferred method the nucleic acid detection oligomerfurther comprises a 2′-O-methyl linkages, a chemiluminescent label, anacridinium ester label, a synthetic linker or combinations thereof.

DETAILED DESCRIPTION

The present invention includes methods of detecting HAV present insamples. These samples may be biological samples derived from humans(e.g., feces, blood, serum, saliva or urine), environmental samples(e.g., water, soil) or other materials (e.g., foodstuffs) that arepotentially contaminated with HAV. A sample includes any liquid that maycontain HAV or solid that may contain or have surface HAV. Samplesinclude, for example, those from environmental sources such as water,biological sources such a human fluids or wastes, and food, packagingmaterials, or other components used in food processing. A biologicalsample includes any tissue or material derived from a living or deadhuman which may contain HAV or HAV nucleic acid, including, for example,saliva, blood, plasma, serum, biopsy tissue, gastrointestinal tissue,urine, feces, or other body fluids, tissues or materials. A sample maybe treated to physically or mechanically disrupt its physical state torelease HAV particles or HAV RNA into an aqueous solution or solvent byusing standard methods.

The present invention encompasses nucleic acid compositions, such asoligomers that hybridize specifically to HAV RNA or nucleic acidsderived from HAV RNA, e.g., cDNA or amplified sequences made from HAVRNA. One such composition is a capture oligomer used to purify HAV RNAfrom a complex mixture such as a sample by hybridizing specifically toHAV RNA and attaching the hybridized HAV RNA to a capture support thatpermits separation of the captured HAV RNA from other sample components.The method of purification that uses such a capture oligomer isgenerally referred to as target capture, where HAV RNA is the specifictarget nucleic acid. Another oligomer of the invention is a nucleic acidamplification oligomer (sometimes referred to as a primer). Additionalembodiments include probe oligomers that hybridize specifically to HAVRNA or amplified HAV nucleic acid sequences to provide a signal thatdetects the presence of an HAV specific sequence. These nucleic acidsequences are useful for capturing, amplifying and detecting HAVspecific sequences and, thus, function together for detecting thepresence of HAV in a sample.

The methods are based on detecting the presence of HAV nucleic acidsequences by amplifying in vitro a region of the HAV genome anddetecting the amplified nucleic acid by using a probe that bindsspecifically to a sequence in the amplified nucleic acid. One embodimentof the method includes a step of isolating or purifying HAV nucleic acidfrom a sample before the step of amplifying a region of the HAV genome.This embodiment isolates HAV genomic RNA by using a capture oligomerthat binds specifically to a sequence in the HAV genome, preferablyoutside of the region of the HAV genome that is amplified, andseparating the complex made up of the capture oligomer and the bound HAVRNA from other sample components by using a capture support, such as aparticle to which the capture oligomer also binds. Amplifying a portionof the HAV genomic sequence uses one or more amplification oligomersthat bind specifically to HAV RNA or a complementary sequence, andenzymatic synthesis in vitro to make additional copies of a portion ofthe HAV genomic sequence or a complementary sequence by using theamplification oligomers as primers for synthesis of the additionalcopies. A preferred embodiment uses an isothermal amplification reactionto make additional copies of a portion of the HAV genomic sequence. Theamplified HAV sequence is then detected by specifically binding one ormore probe oligomers to the amplified nucleic acid and detecting asignal that results from the probe oligomer bound to the amplifiedsequence. Detection of a signal resulting from the probe oligomer boundto the amplified HAV sequence indicates the presence of HAV in thesample. These method are useful for detecting the presence of HAV in avariety of samples, such as biological samples used to diagnose a HAVinfection in a human, or HAV-contaminated environmental samples toprevent the spread of HAV resulting from use or consumption of thecontaminated source. These methods are also useful for testing humanfluid samples for the presence of HAV, such as in serum or plasma, toprevent subsequent HAV infections resulting from use of the human fluidin transfusion or for preparation of therapeutic factors. The methods ofthe present invention are also useful for screening human tissue ororgans for the presence of HAV to prevent their use in transplantationtherapy. Thus, these methods are especially important for detecting HAVcontamination in human samples or products derived from human tissue.

Nucleic acids include DNA or an analog thereof, RNA or an analogthereof, or mixed DNA-RNA polymers or oligomers, made up of at leasttwo, and preferably ten or more bases linked by a backbone structure.DNA and RNA may be made up of the common bases (A, T, G and C for DNA,and A, G, C and U for RNA), although base analogs (e.g., inosine) andabasic positions (i.e., a phosphodiester backbone that lacks anucleotide at one or more positions, see U.S. Pat. No. 5,585,481) arealso included in these terms. Polymers may be many hundreds or thousandsof nucleotides long, whereas oligomers generally refer to nucleic acidsof 1,000 or fewer linked nucleotides, and often comprise two to about100 linked nucleotides. Oligomers generally fall in a size range havinga lower limit of about 10 bases and an upper limit of about 150 bases,preferably in a size range of about 15 to about 70 bases. Oligomers maybe purified from naturally occurring biological sources, but preferablyare synthesized in vitro using any of a variety of well-known enzymaticor chemical methods (e.g., Caruthers et al., 1987, Methods in Enzymol.154: 287).

A nucleic acid backbone refers to groups or linkages known in the art(Eschenmoser, 1999, Science 284:2118-2124), e.g., sugar-phosphodiesterlinkages, 2′-O-methyl linkages, guanidine linkers in DNA (“DNG”),S-methylthiourea linkers, methylphosphonate linkages, phosphoramidatelinkages, amide backbone modifications as in polyamide or peptidenucleic acids (PNA), phosphorothioate linkages, phosphonic ester nucleicacid linkages, pyranosyl oligonucleotide linkages, bicyclo- andtricyclo-nucleic acid linkages, formacetal and 3′-thioformacetallinkages, morpholino linkages, or other modifications of the naturalphosphodiester internucleoside bond, or combinations thereof (Majlessiet al., 1998, Nucl. Acids Res. 26(9):2224-2229; Dempcy et al., 1995,Proc. Natl. Acad. Sci. USA 92:6097-6101; Browne et al., 1995, Proc.Natl. Acad. Sci. USA 92:7051-7055; Arya & Bruice, 1998, J. Am. Chem.Soc. 120:6619-6620; Reynolds et al., 1996, Nucl. Acids Res.24(22):4584-4591; Gryaznov & Chen, 1994, Am. Chem. Soc. 116:3143-3144;Chaturvedi et al., 1996, Nucl. Acids Res. 24(12):2318-2323; Hyrup &Nielsen, 1996, Bioorg. & Med. Chem. 4:5-23; Hydig-Hielsen et al., PCTPat. App. WO 95/32305; Mesmaeker et al., Syn. Lett., November1997:1287-1290; Peyman et al., 1996, Angew. Chem. Int. Ed. Engl.35(22):2636-2638; Aerschot et al., 1995, Angew. Chem. Int. Ed. Engl.34(12):1338-1339; Koshkin et al., 1998, J. Am. Chem. Soc.120:13252-13253; Steffens & Leumann, 1997, J. Am. Chem. Soc.119:11548-11549; Jones et al., 1993, J. Org. Chem. 58:2983-2991;Summerton & Weller, 1997, Antisense & Nucl. Acid Drug Dev. 7:187-195;Stirchak et al., 1989, Nucl. Acids Res. 17(15):6129-6141). A nucleicacid backbone may include a mixture of linkages in the same oligomer orpolymer (e.g., one or more sugar-phosphodiester linkages and one or more2′-O-methyl linkages in the strand) or may have the same linkagesthroughout the strand (e.g., all 2′-O-methyl or all amide modificationlinkages).

A target, target sequence, or target nucleic acid may refer to a largesequence (e.g., greater than 1000 nt) or a smaller sequence within alarger nucleic acid, to which another sequence binds, e.g., by usingstandard complementary base pairing. A target nucleic acid may be RNA orDNA, which is naturally occurring or made synthetically. For example, atarget may be a relatively large nucleic acid such as the HAV genome, ora target may be a smaller subsequence contained in HAV RNA, itscomplement, or an amplification product made from it, which bindsspecifically another sequence in an oligomer. Those skilled in the artwill appreciate that a target nucleic acid may exist in any form, e.g.,a sense or antisense (+ or −) strand.

Complementary nucleic acids (or nucleic acid complementarity) refers toa base sequence in one strand of nucleic acid that, due to orientationof its functional groups, binds to a base sequence in an opposingstrand, e.g., by hydrogen bonding between A and T or U bases, andbetween C and G bases. Substantially complementary means that a basesequence in one strand is not completely or perfectly complementary to abase sequence in an opposing strand, but that sufficient bonding occursbetween bases of the two strands to form a stable hybridized complex ina set of conditions (e.g., salt concentration in an aqueous solution, ora temperature). Such conditions may be predicted by using the basesequences and standard mathematical calculations known to those skilledin the art for determining the melting temperature (Tm) at which 50% ofhybridized strands are denatured, or by empirical determination of Tm byusing routine methods (e.g., see Sambrook et al., Molecular Cloning, ALaboratory Manual, 2nd Ed., (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989), at 9.50-51, 11.46-49, 11.55-57).

A hybridization condition refers to the cumulative environment in whichone nucleic acid strand bonds to a second nucleic acid strand bycomplementary strand interactions to produce a hybridization complex.Such conditions include, e.g., temperature, chemical components andconcentrations of compounds (e.g., salts, buffers, chelating agents,organic compounds) in aqueous and/or organic solutions that contain thenucleic acids. Other factors, such incubation time or reaction chamberdimensions may contribute to hybridization conditions, which are wellknown in the art (e.g., see Sambrook et al., Id., at 1.90-1.91,9.47-9.51, 11.47-11.57).

A label refers to a molecular moiety that is detectable or produces adetectable response directly or indirectly, e.g., by catalyzing areaction that produces a signal. Labels include luminescent moieties(e.g., fluorescent, bioluminescent, or chemiluminescent compounds),radioisotopes, members of binding pairs (e.g., biotin and avidin orstreptavidin), enzymes or enzyme substrates, reactive groups orchromophores, e.g., a dye or particle that results in a detectablecolor. A detectable response or signal is any perceptible or measurableoutput that indicates the presence of a label, e.g., light, color,radioactive decay emission, electrical signal, magnetic field, or signalblockage, such as from quenching or turbidity.

An immobilized oligomer or probe refers to an oligomer that is connectedor attached, covalently or noncovalently, to a capture support matrix,which provides a means for joining a capture hybrid containing a targetnucleic acid to the capture support. A preferred immobilized probe is anoligomer that binds, directly or indirectly, to a target nucleic acid tofacilitate separation of the bound target nucleic acid from unboundsample materials. In one embodiment, the target is indirectly bound tothe immobilized probe via a capture probe that links the target andimmobilized probe in a hybridization complex (see U.S. Pat. Nos.6,110,678 and 6,280,952, Weisburg et al.). Any of a variety of supportsmay be used, such as matrices or particles made of, e.g.,nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene,silane polypropylene, and magnetic materials. Monodisperse magneticparticles of relatively uniform size that can be readily retrieved fromsolution by applying a magnetic force are a preferred embodiment of asupport.

A capture oligomer or probe joins a target nucleic acid and animmobilized probe, i.e., by using a target-specific moiety that bindsthe target sequence and a moiety that attaches the capture probe to animmobilized probe. In one embodiment, both attachments result fromhybridization of complementary base sequences, i.e., hybridization of atarget sequence with a target-complementary sequence of the captureprobe, and hybridization of another portion of the capture probe to acomplementary sequence of the immobilized probe. In other embodiments,one or more attachments may occur by using members of a specific bindingpair (e.g., biotin and avidin or streptavidin), which are well-known inthe art. Compositions and methods that use capture probes are known(U.S. Pat. No. 6,110,678).

Separating or purifying refers to removing one or more components of asample from other sample components. Sample components include nucleicacids in a generally aqueous solution phase, which may also includematerials such as proteins, carbohydrates, lipids, and other compounds.Preferably, separating or purifying a nucleic acid removes at leastabout 70%, more preferably at least about 90% and, even more preferably,at least about 95% of the nucleic acid from other sample components.

An amplification oligonucleotide or oligomer refers to an oligomer thathybridizes to a target nucleic acid, or its complementary sequence, andparticipates in a nucleic acid amplification reaction by serving as aprimer for synthesis of nucleic acid in vitro. Amplification oligomersmay contain additional functional sequences, such a promoter sequencethat binds an RNA polymerase in an oligomer referred to as a promoterprimer. An amplification oligonucleotide generally contains at leastabout 10 contiguous bases, and preferably at least about 12 contiguousbases, that are complementary to a target sequence (or a complementarystrand thereof). The contiguous bases are preferably at least about 80%,more preferably at least about 90%, and most preferably about 100%complementary to the sequence that binds to the amplification oligomer.An amplification oligomer may be RNA, DNA, or mixed DNA-RNA bases, andoptionally may include modified nucleotides or backbone linkages.

A primer refers to an oligonucleotide that hybridizes to a templatenucleic acid and which has an end (usually 3′) that can be extended in apolymerization reaction catalyzed by an enzyme. The 5′ region of theprimer may be non-complementary to the target nucleic acid, e.g., as ina promoter primer that includes a 5′ promoter sequence that is notpresent in the target sequence. Those skilled in the art will appreciatethat a promoter primer may function as a primer independent of itspromoter sequence (i.e., with or without the promoter sequence) and thatany amplification oligomer may be modified to include a 5′ promotersequence, and thus function as a promoter primer.

Amplification refers to any known procedure for obtaining multiplecopies of a target sequence, its complement, or fragments thereof.Amplification of fragments refers to production of an amplified nucleicacid that contains less than the complete target nucleic acid sequenceor its complement, e.g., amplification of a portion of the complete HAVgenome. Amplification of a fragment or portion of the complete targetmay result from using an amplification oligomer that which hybridizesto, and initiates polymerization from an internal position of the targetnucleic acid. Known amplification methods include, e.g.,transcription-mediated amplification (TMA), replicase-mediatedamplification, the polymerase chain reaction (PCR), ligase chainreaction (LCR) and strand-displacement amplification (SDA).Replicase-mediated amplification uses self-replicating RNA molecules,and a replicase such as QB-replicase (e.g., U.S. Pat. No. 4,786,600Kramer et al.). PCR uses a DNA polymerase, multiple primers and thermalcycling to synthesize many copies of two complementary strands of DNA orcDNA (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, Mulliset al.). LCR uses at least four separate oligomers to amplify a targetand its complementary strand by using multiple cycles of hybridization,ligation, and denaturation (e.g., U.S. Pat. No. 5,427,930 Biekenmeyer etal., and U.S. Pat. No. 5,494,810 Barany et al.). SDA uses a primer thatcontains a recognition site for a restriction endonuclease and theendonuclease nicks one strand of a hemimodified DNA duplex that includesthe target sequence, followed by a series of primer extension and stranddisplacement steps (e.g., U.S. Pat. No. 5,422,252 Walker et al.)Transcription-mediated or transcription-associated amplificationreactions use a polymerase to make a complementary strand to the targetin a double-stranded form that contains a functional promoter for aspecific RNA polymerase that makes transcripts that can cycleisothermically to produce additional copies of transcripts that aredetectable amplification products.

Transcription-mediated or transcription-associated amplification uses anRNA polymerase to produce multiple RNA transcripts from a nucleic acidtemplate in isothermal reactions that use an RNA polymerase, a DNApolymerase, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, and a promoter-primer, and optionally may include one ormore additional oligonucleotides. These methods of amplification andreaction conditions have been described in detail previously (e.g., seeU.S. Pat. Nos. 5,399,491 and 5,554,516 Kacian et al., U.S. Pat. No.5,437,990 Burg et al., PCT Nos. WO 88/01302 and WO 88/10315 Gingeras etal., U.S. Pat. No. 5,130,238 Malek et al., U.S. Pat. Nos. 4,868,105 and5,124,246 Urdea et al.).

Preferred embodiments of the present invention usetranscription-mediated amplification (TMA, described in U.S. Pat. Nos.5,399,491 and 5,554,516). It will, however, be apparent to one skilledin the art that the methods and oligonucleotide primer sequencesdescribed herein are readily applicable to use with any nucleic acidamplification method that synthetically extends primers by using apolymerase.

A detection probe is an oligomer that binds to a specific targetsequence and, by binding, produces, directly or indirectly, a detectablesignal that indicates the presence of the target sequence. A detectionprobe need not be labeled to produce a detectable signal, an example ofsuch is an electrical impulse that results from the probe binding to thetarget. A labeled probe is made up of an oligomer that is linked,directly or indirectly, to a label. Methods of making and/or usinglabeled probes are well known (e.g., Sambrook et al., id., Chapt. 10;U.S. Pat. No. 6,361,945 Becker et al., U.S. Pat. No. 5,658,737 Nelson etal., U.S. Pat. No. 5,656,207 Woodhead et al., U.S. Pat. No. 5,547,842Hogan et al., U.S. Pat. No. 5,283,174 Arnold et al., U.S. Pat. No.4,581,333 Kourilsky et al., and U.S. Pat. No. 5,731,148 Becker et al.).Detection probes may include a synthetic linker (U.S. Pat. Nos.5,585,481 and 5,639,604 Arnold et al.), and a chemiluminescent label,such as an acridinium ester (AE) compound (U.S. Pat. Nos. 5,185,439,5,656,207, and 5,658,737).

A homogeneous detectable label is a label that can be detected in ahomogeneous manner depending on whether the label is bound or unbound toa target. That is, detection of a label in a homogeneous reaction doesnot require physical separation of unbound forms of the label from themixture in which the signal is detected. It will be appreciated byskilled artisans that a homogeneous reaction may occur in solution or ona support, e.g. on an array, biochip or gene chip. Homogeneousdetectable labels and conditions for their detection are well known(e.g., U.S. Pat. Nos. 5,283,174, 5,656,207 and 5,658,737).

By “consisting essentially of” is meant that additional component(s),composition(s) or method step(s) that do not materially change the basicand novel characteristics of the present invention may be included inthe compositions, kits, or methods of the present invention. Suchcharacteristics include the ability to detect specifically the presenceof HAV nucleic acid in a sample with a sensitivity of at least 80% forsamples containing 25 to 30 copies of HAV per ml by using a combinationof capture probe, amplification primers, and detection probe oligomersas described herein. Any component(s), composition(s), or method step(s)that have a material effect on the specificity and/or sensitivity ofdetection of HAV present in a sample by using the nucleic acid oligomersand in vitro methods described herein, would fall outside of this term.

Unless defined otherwise, all scientific and technical terms used hereinhave the same meaning as commonly understood by those skilled in therelevant art. Definitions of many of the terms used herein are providedin, e.g., Dictionary of Microbiology and Molecular Biology, 2nd ed.(Singleton et al., 1994, John Wiley & Sons, New York, N.Y.), TheEncyclopedia of Molecular Biology (Kendrew, Ed., 1994, Blackwell ScienceLtd., Cambridge, Mass.), or The Harper Collins Dictionary of Biology(Hale & Marham, 1991, Harper Perennial, New York, N.Y.). Unlessmentioned otherwise, the techniques employed or contemplated herein arestandard methodologies well known to one of ordinary skill in the art.Examples are included to illustrate some embodiments of the invention.

The present invention includes compositions (nucleic acid amplificationoligomers, detection probes, and optionally capture oligomers) andmethods for detecting HAV nucleic acid in a sample. To select sequencesappropriate for use as the oligomers disclosed herein, known HAV genomicsequences (Beneduce, et al., 1995, Virus Res. 36 (2-3): 299-309,Fujiwara, et al., 2001, J. Hepatol. 35 (1): 112-119, Hu, et al., 2002,Acta Virol. 46 (3): 153-157), including those of different isolates,partial sequences, and complementary sequences available on a publicdatabase (e.g., GenBank accession nos. AB020564 to AB020569) werealigned by matching regions of the same or similar sequences and thealigned sequences were compared using well known techniques. Althoughsequence comparisons may be facilitated by use of algorithms, thoseskilled in the art can readily perform such comparisons manually andvisually. Portions of HAV sequences that contain relatively few sequencevariants between the compared sequences were chosen as a basis fordesigning synthetic oligomers suitable for use in the capture,amplification and detection steps described herein. Other well-knownsequence characteristics, such as the GC content and the relativeabundance of predicted secondary structures (e.g., hairpin turns orintramolecular pairing), were also considered in selecting the oligomersequences.

Based on these analyses, regions of the HAV genome around nucleotides200, 3700, 4700, 5700, 5800, 6000, and 7000 were chosen as potentialtarget regions for detection of amplified HAV sequences. For eachregion, oligomers were designed for use in capturing the HAV RNA from asample to purify it from other sample components, as amplificationoligomers, and as probe sequences. Preferred embodiments of targetregions are in portions of 0 to 305 nt, 4714 to 4765 nt, 5495 to 5788nt, 5788 to 6069 nt, and 6952 to 7413 nt of the HAV genome.

Capture oligomer sequences generally include a sequence that bindsspecifically to a sequence near the target region to be amplified and a“tail” region used in attaching the hybridization complex that includesthe target to a solid support, for example via hybridization to animmobilized oligomer (e.g., U.S. Pat. No. 6,110,678). Preferred captureoligomers include a target-specific sequence that binds specifically toa HAV RNA sequence and a covalently attached tail sequence (e.g.,dT.sub.3dA.sub.30), as shown in SEQ ID NOS:1 to 7. Those skilled in theart will understand that the target-specific portion of a captureoligomer (SEQ ID NOS:8 to 14) or its RNA equivalent may be linked to anymoiety that allows it to bind to an immobilized probe (e.g., a differenttail sequence or a member of a binding pair, such as biotin or avidin).Any backbone may link the base sequence of a capture oligomer. Someembodiments use 2′-O-methyl linkages in the target-specific portion of acapture oligomer and standard DNA linkages in the tail portion. Apolynucleotide tail sequence may be any sequence complementary to asequence of an immobilized probe, and generally has a sequence length ofabout 5 to 50 residues, and is preferably a substantially homopolymericsequence in a range of about 10 to about 40 residues (e.g., C.sub.10 toC.sub.40) that is complementary to an immobilized homopolymeric sequence(e.g., G.sub.15).

Primer sequences bind specifically to an HAV RNA target sequence or acomplementary strand and flank a target sequence that is amplified,although primer sequences may contain additional sequences that do notbind to the target or its complementary sequence. A primer may be apromoter primer and include a 5′ promoter sequence, such as a T7 RNApolymerase promoter (SEQ ID NO:19). Embodiments of promoter primersinclude those of SEQ ID NOS:20 to 49. Other embodiments of HAV-specificprimers may include ancillary sequences, such as restrictionendonuclease recognition sequences (SEQ ID NOS:132 to 135). Thoseskilled in the art will appreciate that a target-specific sequence of aprimer, with or without an attached promoter or ancillary sequence, mayserve as a primer in a variety of in vitro amplification conditions.Amplification oligomers were designed for sequences in targeted regionsof the HAV genome (e.g., around nucleotide positions 200, 3700, 4700,5700, 5800, 6000, and 7000). Those skilled in the art will appreciatethat these numbers refer to HAV target regions that are approximate onlyand that oligomers may function in an assay for more than one targetregion. That is, the identifying target region numbers provided heremerely as a convenient shorthand reference for grouping preferredembodiments of the invention does not functionally restrict theseoligomers. Amplification oligomers may be synthesized as DNA, RNA,complementary DNA or RNA sequences, or mixed DNA and RNA sequences, andmay include one or more non-standard nucleic acid backbone linkages. Forexample, an oligomer of SEQ ID NO:106 was synthesized with RNA bases and2′-O-methyl linkages at residues 1 to 4 and standard DNA bases andlinkages at the other residues.

For a first HAV target region (around position 200), amplificationoligomers include those in a size range of about 23 to 26 nt that arecontained in the sequence of SEQ ID NO:138, and include at least thesequence of SEQ ID NO:139 or SEQ ID NO:140. Embodiments of sucholigomers includes those of SEQ ID NO:51 to SEQ ID NO:57. Embodiments ofpromoter primers for this region, in a size range of about 50 to 53 nt,are those that include target-specific portions of SEQ ID NOS:21 to 27.Amplification oligomers for this target region also include those in asize range of about 19 to 25 nt contained in SEQ ID NO: 141, and containat least the sequence of any one of SEQ ID NOS:142 to 146. Embodimentsof amplification oligomers for this target region include those of SEQID NOS:15 to 18, 20 to 27, 50 to 57, and 80 to 85.

For a second HAV target region (around position 3700), amplificationoligomers include those in a size range of about 21 to 27 nt, containedin the sequence of SEQ ID NO:60, or in SEQ ID NO:86 and include at leastSEQ ID NO:156. Embodiments of promoter primers that include suchtarget-specific portions for this region and are in a size range ofabout 48 to 54 nt include those of SEQ ID NOS:29 to 32. Embodiments ofamplification oligomers for this target region include those of SEQ IDNOS:28 to 30, 58 to 60, and 86 to 88.

For a third HAV target region (around position 4700), amplificationoligomers include those in a size range of about 24 to 30 nt that arecontained in SEQ ID NO:147 and include at least the sequence of SEQ IDNO:148, or are contained in SEQ ID NO:157 and include at least thesequence of SEQ ID NO:158. Embodiments of amplification oligomers forthis target region include those of SEQ ID NOS:31, 32, 61, 62, 89, 90,and 91, of which SEQ ID NO:31 and SEQ ID NO:32 are promoter-primerembodiments that include a 5′ promoter sequence attached to thetarget-specific sequence.

For a fourth HAV target region (around position 5700), amplificationoligomers include those in a size range of about 18 to 27 nt that arecontained in the sequence of SEQ ID NO:93 or SEQ ID NO:95. Embodimentsof such oligomers include those that contain at least any one of SEQ IDNO:97, SEQ ID NO:159, and SEQ ID NO:160. Embodiments of amplificationoligomers for this target region include those of SEQ ID NOS:33, 63, and92 to 97, of which SEQ ID NO:33 is a promoter primer embodiment thatincludes a 5′ promoter sequence attached to the target-specificsequence.

For a fifth HAV target region (around position 5800), amplificationoligomers include those in a size range of about 19 to 31 nt that arecontained in SEQ ID NO:149 and include at least the sequence of SEQ IDNO:150. Embodiments of promoter primers, in a size range of about 51 to56 nt, that include such target-specific portions are those of SEQ IDNOS:34 to 40. Other embodiments of amplification oligomers for thistarget region include those of SEQ ID NOS:64 to 70, and 97.

For a sixth HAV target region (around position 6000), amplificationoligomers include those of about 24 to 28 nt contained in the sequenceof SEQ ID NO:161 and include the sequence of SEQ ID NO:162. Embodimentsof amplification oligomers for this target region include those of SEQID NOS:41, 42, 71, 72, 98, 99, and 101, of which SEQ ID NOS:41 and 42are embodiments of promoter primers that include a 5′ promoter sequenceattached to the target-specific sequence.

For a seventh HAV target region (around position 7000), amplificationoligomers include those in a size range of about 20 to 30 nt containedin SEQ ID NO: 151 and that include at least any one of the sequences ofSEQ ID NO:152 to SEQ ID NO:155. Other embodiments of amplificationoligomers for this target region are contained in SEQ ID NO:163 andinclude at least the sequence of SEQ ID NO:164. Additional embodimentsare amplification oligomers that are contained in SEQ ID NO:165 andinclude at least any one of the sequences of SEQ ID NOS:166 to 168.Embodiments of promoter primers, in a size range of about 51 to 56 nt,that include HAV target-specific portions for this region are SEQ IDNOS:43 to 49. Other embodiments of amplification oligomers for thisregion include those of SEQ ID NOS:73 to 79, and 102 to 108.

Oligomers were designed to hybridize to and detect amplified HAVsequences, which include the detection probes of SEQ ID NOS:109, 111,113, 115, 117, 119, 121, 122, 123, 124, and 126 to 130. Those skilled inthe art will appreciate that a detection probe will be chosen tohybridize to a sequence contained within an amplified sequence that isdetermined by the combination of amplification oligomers that are used.Detection probe oligomers may be synthesized as DNA, RNA, or mixed DNAand RNA polymers, and may include alternative backbone linkages such as2′-O-methyl linkages. For example, oligomers of SEQ ID NOS:109, 111,117, 119, 121, 122, 128, and 130 were synthesized with 2′-O-methyllinkages, and oligomers of SEQ ID NOS:124 and 127 were synthesized asmixed DNA and RNA nucleotides with 2′-O-methyl linkages from the secondresidue to the 3′ terminal residue. Preferred embodiments of detectionprobes have an attached chemiluminescent label, preferably an acridiniumester (AE) compound (U.S. Pat. Nos. 5,185,439, 5,639,604, 5,585,481, and5,656,744), which in preferred embodiments are attached to the probe bya non-nucleotide linker (see U.S. Pat. Nos. 5,585,481, 5,656,744, and5,639,604, particularly at column 10, line 6 to column 11, line 3, andExample 8). Embodiments of the probe oligomers were labeled by usingknown methods with an AE compound between residues 9 and 10 for SEQ IDNOS:119, 121 and 124, between residues 10 and 11 for SEQ ID NOS:115,117, 126, 127 and 128, between residues 11 and 12 for SEQ ID NOS:109,111, 123, 124 and 130, between residues 12 and 13 for SEQ ID NOS:113,122 and 129, and between residues 13 and 14 for SEQ ID NO:122. Probeoligomers were tested and characterized by hybridization withcomplementary oligomer sequences, by using standard methods fordetermining Tm and/or differential hydrolysis of the acridinium ester ina hybridization complex (described in detail in U.S. Pat. No.5,283,174). For example, hybridizations were performed by usingcomplementary pairs of sequences of SEQ ID NO:109 and SEQ ID NO:110, SEQID NO:111 and SEQ ID NO:112, SEQ ID NO:113 and SEQ ID NO:114, SEQ IDNO:115 and SEQ ID NO:116, SEQ ID NO:117 and SEQ ID NO:118, SEQ ID NO:119and SEQ ID NO:120, SEQ ID NO:124 and SEQ ID NO:125, SEQ ID NO:128 andSEQ ID NO:100, and SEQ ID NO:130 and SEQ ID NO:131. Hybridizationtesting may be performed by using other complementary sequences, such asSEQ ID NO:124 with SEQ ID NO:137, and SEQ ID NO:129 with SEQ ID NO:136.

Compositions of the present invention include kits for detecting HAVnucleic acid sequences. Such kits include amplification oligomers asdisclosed herein that function as primers for amplifying HAV nucleicacid sequences in vitro. Exemplary kits include a first amplificationoligomer that hybridizes specifically to a sequence in a target regionof the HAV RNA genome or its complementary sequence, and a secondamplification oligomer that hybridizes specifically to another HAVsequence in the target region, preferably one complementary to the HAVRNA genomic sequence. Embodiments of kits include amplificationoligomers that are combinations of primers and promoter primers asdescribed herein. Kits may also contain one or more oligomers that serveas detection probes for detecting amplified HAV sequences of the targetregion of the primers selected for the kit. Embodiments of kits thatinclude of probe oligomers use one or more of the detection probesequences as described herein, which may include a label attacheddirectly or indirectly to the probe oligomer. Kits may also containoligomers that serve as capture oligomers for purifying a target HAV RNAfrom a sample. Embodiments of such capture oligomers as described hereinmay contain a covalently attached tail sequence or other binding moietyused in target capture. Kits useful for practicing the methods describedherein are also included in the invention, and preferred embodimentsinclude at least two amplification oligomers as described herein, andmay also include reagents for performing in vitro amplification, e.g.,enzymes, salt solutions, and nucleic acid synthesis substrate compounds.Oligomers described herein may be packaged in a variety of differentembodiments, and thus, those skilled in the art will appreciate that theinvention embraces many different kit configurations. For example, a kitmay include amplification oligomers for only one target region of theHAV genome, or it may include amplification oligomers for multiple HAVtarget regions. Those skilled in the art will appreciate that a kit thatincludes a detection probe will include a probe that binds to a sequenceamplified by the amplification oligomers of the kit. That is, selectionof amplification oligomers and detection probe oligomers for a kit willbe linked by their intended target region.

An embodiment of the assay to detect HAV nucleic acid in a sampleincludes the steps of capturing the HAV target nucleic acid from asample by using a capture oligomer, amplifying a region of the capturedHAV nucleic acid by using a combination of at least two primers, anddetecting the amplified HAV sequence by specifically hybridizing it witha detection probe oligomer and detecting a signal that results from theprobe bound to the amplified HAV sequence. Preferred embodiments use atranscription-associated or transcription-mediated amplificationreaction. Either the amplified nucleic acid or the probe may be labeled,or both may be unlabeled and a detectable signal results from anindirect label or response associated with the hybridization complex,such as an electrical impulse resulting from hybridization of the probeand the amplified nucleic acid.

The capturing step preferably uses a capture oligomer that includes atarget-specific sequence (e.g., SEQ ID NOS:8 to 14) that specificallyhybridizes to a HAV target sequence and a moiety that permits thehybridized target nucleic acid to be separated from other samplecomponents. The capturing step may use a capture oligomer that alsoincludes a tail portion, e.g. as in SEQ ID NOS:1 to 7, that serve as themoiety that allows the target nucleic acid to be separated from othersample components by hybridization of the tail portion to an immobilizedprobe, as previously described (U.S. Pat. No. 6,110,678). Preferredembodiments use supports that are magnetic spheres that are monodisperse(i.e., uniform in size±about 5%) with covalently attached or immobilizedpoly-dT oligomers that hybridize to a complementary tail sequence of thecapture oligomer. The hybridization complex that includes at least thetarget nucleic acid and the capture oligomer, and preferably alsoincludes the immobilized probe, is separated from the other samplecomponents by using standard physical separation methods (e.g.,application of magnetic force, filtration, or centrifugation) and thecaptured target nucleic acid may be washed one or more times to furtherpurify the target nucleic acid from other sample components. Forexample, particles with the attached target nucleic acid in ahybridization complex are suspended one or more times in a washingsolution that maintains the complex and then the particles with theattached complex are retrieved from the washing solution as describedabove.

Amplifying the captured HAV target sequence uses an in vitroamplification reaction that uses at least two primers that flank thesequence to be amplified, e.g., an HAV sequence flanked by SEQ ID NO:66and SEQ ID NO:95, or their complementary sequences. One embodiment usesa transcription-associated amplification reaction that makes many RNAcopies of a sequence in substantially isothermal conditions (asdescribed previously in U.S. Pat. Nos. 5,399,491 and 5,554,516).Transcription-associated amplification uses two types of primers (one apromoter primer that contains a promoter sequence for an RNApolymerase), enzymes (a reverse transcriptase and an RNA polymerase),substrates (deoxyribonucleoside triphosphates, ribonucleosidetriphosphates) and appropriate salts and buffers in solution to producemultiple RNA transcripts from a nucleic acid template. Briefly, apromoter primer hybridizes specifically to a target RNA sequence andreverse transcriptase creates a first strand cDNA by extension from the3′ end of the promoter primer and degrades the template strand in theresulting DNA:RNA duplex by using RNase H activity. A second primerbinds to the cDNA and another DNA strand is synthesized by the reversetranscriptase from the end of the second primer, to create adouble-stranded DNA with a functional promoter sequence to which the RNApolymerase binds. Multiple RNA transcripts (“amplicons”) are transcribedand each can be a template in a new round of replication as describedabove, thus generating large amounts of single-stranded amplifiedsequence (e.g., about 100 to 3,000 transcripts from a single template).Embodiments of the invention that use a transcription-associatedamplification reaction may use promoter primers (SEQ ID NOS:20 to 49)with other primers (SEQ ID NOS:15 to 18, 80 to 99, and 101 to 108) toamplify selected HAV sequences for detection.

The detecting step uses at least one probe that binds specifically tothe amplified HAV sequences. Embodiments may use any know detectionmethod (e.g., detection of a radioactive, fluorescent, enzymatic,colorimetric, electrical, or luminescent signal) to detect binding ofthe detection probe to the amplified HAV sequences, and the detectedsignal indicates the presence of HAV in the sample. Embodiments of probeoligomers (SEQ ID NOS:109, 111, 113, 115, 117, 119, 121, 122 to 124, 126to 130) may be unlabeled or labeled using any of a variety of knownlabels. In preferred embodiments, the detection step is performed in ahomogeneous detection reaction without removing the unbound detectionprobe from the mixture. Embodiments of the probe oligomers for use inhomogeneous detection reactions are preferably labeled with one of avariety of AE compounds, which produce a chemiluminescent signal that isdetected as described in detail previously (U.S. Pat. Nos. 5,283,174,5,656,744, and 5,658,737).

A preferred assay embodiment generally includes the following steps. AHAV-containing sample is provided, which may be prepared by usingstandard laboratory methods to make a substantially aqueous solution orsuspension that contains HAV. An aliquot (0.5 ml) of the sample solutionor suspension is mixed with about an equal volume (0.4 to 0.5 ml) of atarget capture reagent, i.e., a solution that contains one or morecapture oligomers (4 pmol/reaction), magnetic particles with attachedimmobilized probes complementary to a portion of the capture oligomers,and salt compounds to provide a hybridization condition. The targetcapture reagent preferably includes a detergent or other chaotropicagent that disrupts the HAV particles and releases HAV RNA forhybridization with capture oligomers. The mixture is incubated 20-30 minat 60.deg.C. to allow hybridization of the target-specific portion ofthe capture oligomer to the HAV target sequence and then at roomtemperature for 20-30 min to allow binding of the capture oligomer andimmobilized probe. A magnetic field is applied to the outside of thereaction container for about 10 min to separate the particles with theattached hybridization complexes that include HAV RNA, and the solutionphase containing other sample components is aspirated away. To wash theparticles with attached hybridization complexes, they are suspended in 1ml of a wash buffer, separated from the solution substantially asdescribed above, and the solution is removed. Particles with attachedhybridization complexes that include the purified HAV RNA are mixed witha solution that contains amplification reagents (buffers, salts, dXTPand XTP substrates), and a combination of amplification oligomers (apromoter primer and a primer combination, each at 3 to 30 pmol,generally 15 pmol each), and covered with oil (0.2 ml of filteredsilicon oil) to prevent evaporation, and incubated for 10 min at60.deg.C., then for 10 min at 42.deg.C., and then enzymes are added(reverse transcriptase and RNA polymerase), and the mixture is incubatedfor 60 min at 42.deg.C. For detection, the amplification reactionmixture is incubated with at least one acridinium labeled detectionprobe oligomer to provide a maximum detectable signal (relative lightunits or RLU) of 2 million or less, as detected by using standardmethods on a luminometer (e.g., Gen-Probe Leader®, Gen-ProbeIncorporated, San Diego, Calif.). Detection probe is mixed withundiluted or a diluted aliquot of the amplification reaction mixture ina hybridization solution, incubated for 20 min at 60.deg.C. to allowhybridization of the probe oligomer to the amplified target sequence.Then, label on unbound probes is hydrolyzed by using a selection reagent(e.g., a base) and incubated for 10 min at 60.deg.C., followed by addinga detection reagent (e.g., H.sub.2O.sub.2) to produce chemiluminescence,followed by pH neutralization (e.g., by adding acid), and detecting thechemiluminescent signal (RLU) on a luminometer (e.g., 1-5 sec).

For use in the methods described above, capture oligomers, amplificationoligomers and detection probes may be synthesized using standard methodsto produce DNA, RNA, or mixed DNA and RNA polymers. Such oligomers mayinclude standard or modified linkages and/or naturally occurringnucleosides (A, T or U, G, C), analogs (e.g., inosine), or syntheticpurine and pyrimidine derivatives (e.g., P or K bases) (Lin & Brown,1989, Nucl. Acids Res. 17:10373-83; Lin & Brown, 1992, Nucl. Acids Res.20: 5149-52).

The general principles of the present invention may be more fullyappreciated by reference to the following examples describe someembodiments of the present invention. In addition to the specificcomponents described in the examples, generally the following reagentswere used in the experiments described below. Target capture reagent wasmade up of 790 mM HEPES, 680 mM LiOH, 10% (v/v) lithium lauryl sulfate(LLS), 230 mM succinic acid, 0.03% (v/v) anti-foaming agent,100.micro.g/ml magnetic particles (1 micron SERA-MAG.sup.™ particles,Seradyn, Inc.

Indianapolis, Ind.) with covalently attached poly-dT.sub.14, and one ormore capture oligomers, each at 4 pmol per 400.micro.l. Wash buffer wasmade up of 150 mM NaCl, 10 mM HEPES, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v/v)ethanol, 0.1% SDS, 0.02% (w/v) methyl paraben, 0.01% (w/v) propylparaben, at pH 7.5. Amplification reagent was made up of 11.6 mM Trisbase, 15 mM Tris-HCl, 22.7 mM MgCl.sub.2, 23.3 mM KCl, 3.33% glycerol,0.05 mM Zn-acetate, 0.665 mM dATP, 0.665 mM dCTP, 0.665 mM dGTP, 0.665mM dTTP, 5.32 mM ATP, 5.32 mM CTP, 5.32 mM GTP, and 5.32 mM UTP, at pH7. Enzyme reagent was made up of 140 U/.micro.l T7 RNA polymerase, 224RTU/.micro.l of Moloney Murine Leukemia Virus reverse transcriptase(MMLV-RT), 16 mM HEPES, 70 mM N-acetyl-L-cysteine, 3 mM EDTA, 0.05%(w/v) Na-azide, 20 mM Tris, 50 mM KCl, 20% (v/v) glycerol, 10% (v/v)TRITON® X-102, 150 mM trehalose, at pH 7. (Enzyme units typically are 1U of T7 RNA polymerase incorporates 1 nmol of ATP into RNA in 1 hr at37.deg.C. using a DNA template containing a T7 promoter, and 1 U ofMMLV-RT incorporates 1 nmol of dTTP in 10 min at 37.deg.C. using200-400.micro.M oligo-dT primer and poly-A template.) Probe reagent wasmade up of 100 mM succinic acid, 2% (w/v) LLS, 230 mM LiOH, 15 mMAldrithiol-2, 1.2 M LiCl, 20 mM EDTA, 20 mM EGTA, 3% (v/v) ethanol,adjusted to pH 4.7 with LiOH. Selection reagent was made up of 600 mMboric acid, 182 mM NaOH, 1% (v/v) octoxynol (TRITON® X-100), at pH 8.5.Detection reagents were Detect Reagent I, which contained 1 mM nitricacid and 32 mM H.sub.2O.sub.2, and Detect Reagent II (to neutralize pH),which was 1.5 M NaOH (see U.S. Pat. No. 5,283,174 for details).

Example 1 Detection Probe Characterization

Oligomers of SEQ ID NOS:109, 111, 113, 119, 123, 126, and 130 weresynthesized using standard phosphoramidite chemistry (Caruthers et al.,1987, Methods in Enzymol., 154: 287) and an acridinium ester (AE) labelwas attached via a linker by using well-known methods (U.S. Pat. Nos.5,185,439 and 5,283,174), and probes were purified by using routinechromatographic methods (e.g., HPLC). Probes were AE labeled betweenresidues 11 and 12 for SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:123, andSEQ ID NO:130, between residues 12 and 13 for SEQ ID NO:113, betweenresidues 10 and 11 for SEQ ID NO:126, and between residues 9 and 10 forSEQ ID NO:119. To characterize the probe oligomers, each was hybridizedwith a complementary DNA and/or RNA oligomer (e.g., SEQ ID NO:109 withSEQ ID NO:110, SEQ ID NO:111 with SEQ ID NO:112, SEQ ID NO:113 with SEQID NO:114, SEQ ID NO:119 with SEQ ID NO:120, and SEQ ID NO:130 with SEQID NO:131), at temperatures below the predicted Tm of the probe, andthen the Tm was experimentally determined by using standard methods. Thedifferential hydrolysis of the AE label in probes hybridized to acomplementary oligomer compared to AE in unbound probe was alsoexperimentally determined by using standard methods (see U.S. Pat. No.5,283,174). Briefly, the ratio of the time required for half of thesignal to be lost due to AE hydrolysis in the hybrid compared to thetime required for hydrolysis of half of the label in unbound probe wasdetermined. The Tm's were in the range of 59.deg.C. to 66.deg.C. foroligomers of SEQ ID NOS:109, 111, 113, 119 and 130 when hybridized to acomplementary DNA, and Tm's were in the range of 76.deg.C. to 81.deg.C.for oligomers of SEQ ID NOS:109, 111, 123, 126 and 130 when hybridizedto a complementary RNA. The differential hydrolysis ratios were in therange of 12 to 25 for probes of SEQ ID NOS:109, 111, 113, 119 and 130when hybridized to complementary DNA, and the differential hydrolysisratios were in the range of 18 to 104 for probes of SEQ ID NOS:109, 111,123, 126 and 130 when hybridized to complementary RNA. Separately,similar hybridization and differential hydrolysis tests were performedfor probes of SEQ ID NO:121 labeled between residues 9 and 10, SEQ IDNO:122 labeled between residues 13 and 14, SEQ ID NO:124 labeled betweenresidues 9 and 10, and SEQ ID NO:130 labeled between residues 11 and 12,and the differential hydrolysis ratios were in the range of 43 to 190when the probes were hybridized to complementary RNA. These resultsshowed that all of these synthetic probe oligomers hybridizedspecifically to their complementary target sequences and produceddetectable signals useful for specifically detecting amplified HAVsequences.

Example 2 Purification of HAV RNA from Samples

Capture oligomers of SEQ ID NOS:1 to 7, synthesized by using standardphosphoramidite chemistry and purified using standard methods, weretested for their ability to capture HAV RNA released from virus in humanplasma samples. Samples were made by adding HAV particles at knownconcentrations to normal human plasma (0.5 ml) and the samplescontaining HAV (e.g., 500 to 1000 per reaction) were mixed with an equalvolume of the target capture reagent containing each capture oligomerindividually (4 pmol/reaction) and polydT-magnetic particles. Themixtures were incubated for 30 min at 60.deg.C., and then for 30 min atroom temperature to form hybridization complexes that capture HAV RNA tothe particles. The magnetic particles with attached captured HAV RNAwere separated by applying a magnetic field for 10 min to the outside ofthe container, then the solution phase was aspirated away to removeother sample components, and the particles with attached hybridizationcomplexes were washed twice sequentially, each using 1 ml of the washbuffer at room temperature and aspirating the washing solution away fromthe particles. Particles with attached hybridization complexes were thensuspended in probe reagent (0.1 ml) containing a labeled detectionprobe, as described in Example 1, and incubated for 20 min at 60.deg.C.,followed by addition of selection reagent (0.2 ml), mixing andincubation for 10 min at 60.deg.C. Production and detection of thechemiluminescent signal was performed by adding 200.micro.l of a detectreagent I, incubation, and pH neutralization of the mixture by adding200.micro.l of detect reagent II, and measuring RLU by using aluminometer, substantially as described above. For all of the captureoligomers tested, the presence of the HAV RNA in the sample was detectedby detecting a positive signal significantly higher than background (RLUfor a similar sample that contained no HAV). The assays showed littlesignificant performance differences between the capture oligomers.

Example 3 Amplification and Detection of HAV Sequences

HAV samples in normal human plasma were prepared substantially asdescribed in Example 2 and the HAV RNA was captured by using variouscombinations of capture oligomers for assays to amplify and detectselected target regions of the HAV genome. For a target region of 0-305residues of the genome, SEQ ID NOS:2, 3, and 4 were used in the capturestep. For a target region of 4714-4765 residues of the genome, SEQ IDNOS:4, 5, 6, and 7 were used in the capture step. For a target region of5495-5788 residues of the genome, SEQ ID NOS:1 and 6 were used in thecapture step. For a target region of 5788-6069 residues of the genome,SEQ ID NO:2 was used in the capture step. For a target region of6952-7413 residues of the genome, SEQ ID NOS:1, 4, 5 and 7 were used inthe capture step. The capture steps were performed substantially asdescribed in Example 2.

The captured HAV RNA was amplified in reactions substantially asdescribed above that contained different combinations of amplificationoligomers to serve as primers for different target regions in the HAVgenome. The primers used to amplify the target regions were as follows:SEQ ID NO:16 and SEQ ID NO:22 for the 0-305 residues region, SEQ IDNO:89 and SEQ ID NO:32 for the 4714-4765 residues region, SEQ ID NO:92and SEQ ID NO:33 for the 5495-5788 residues region, SEQ ID NO:94 and SEQID NO:37 for the 5788-6069 residues region, and SEQ ID NO:108 and SEQ IDNO:46 for the 6952-7413 residues region. The amplification reactionswere all performed substantially the same as described above. That is,particles with the attached HAV RNA from the target capture step weremixed with amplification reagent and the individual combination ofamplification oligomers described above (generally 15 pmol each), andcovered with silicon oil (0.2 ml) to prevent evaporation, and incubatedfor 10 min at 60.deg.C. and then for 10 min at 42.deg.C. The enzymereagent was added (reverse transcriptase and RNA polymerase), and theamplification reactions were incubated for 60 min at 42.deg.C.

For detection, the amplification mixture was incubated with a labeleddetection probe oligomer that hybridizes specifically to sequencescontained in the amplified region. These included SEQ ID NO:109 or SEQID NO:111 for the 0-305 residues region, SEQ ID NO:115 for the 4714-4765residues region, SEQ ID NO:117 for the 5495-5788 residues region, SEQ IDNO:121 and/or SEQ ID NO:122 for the 5788-6069 residues region, and SEQID NO:129 or SEQ ID NO:130 for the 6952-7413 residues region. The probeswere provided in the probe reagent in an amount previously determinedbased on the specific activity of the labeled probe to produce a maximumdetectable signal of 2 million RLU or less from the hybridized labeledprobe. The probes and amplified sequences were incubated in the probereagent at 55-60.deg.C., and the chemiluminescent signal was producedfrom hybridized probes and detected substantially as describe inExamples 1 and 2. For all of the primer combinations tested with thecaptured HAV RNA, the sensitivity of the amplification assay detectedbetween 400 and 1000 copies of HAV RNA present in the samples.

The various combinations of capture oligomers, amplification oligomersand detection probes used for amplification and detection of selectedHAV target regions in these tests are summarized in Table 1.

TABLE 1 Combinations of Oligomers for Testing for HAV in Samples TargetRegion Capture Amplification (Residues) Oligomer(s) Oligomers DetectionProbe(s)  0-305 SEQ ID NOS: 2, SEQ ID NOS: 16 SEQ ID NO: 109 3 and 4 and22 or 111 4714-4765 SEQ ID NOS: 4, SEQ ID NOS: 32 SEQ ID NO: 115 5, 6and 7 and 89 5495-5788 SEQ ID NOS: 1 SEQ ID NOS: 33 SEQ ID NO: 117 and 6and 92 5788-6069 SEQ ID NO: 2 SEQ ID NOS: 37 SEQ ID NOS: 121 and 94and/or 122 6952-7413 SEQ ID NOS: 1, SEQ ID NOS: 46 SEQ ID NO: 129 4, 5and 7 and 108 or 130

Similar experiments were performed using the different capture oligomers(SEQ ID NOS:1, 2, 3, 4, 5, 6, and 7) separately with HAV-containingsamples, in which the target capture step was performed substantially asdescribed above on nine replicates for each assay condition. For all ofthese tests the target region was residues 5788 to 6069 of HAV, forwhich the same amplification oligomers of SEQ ID NO:36 and SEQ ID NO:96were used in the amplification reactions with the captured HAV RNAperformed as described above, and amplified products were detected bymeasuring chemiluminescence from hybridized detection probe (SEQ IDNO:123 or 124, labeled with AE between residues 11 and 12) as describedabove. The results of these assays are shown in Table 2 (average RLU fornine replicates).

TABLE 2 Amplification and Detection of the 5788-6069 residue TargetRegion Purification by Detected Signal Capture Oligomer (mean RLU) SEQID NO: 1 292,136 SEQ ID NO: 2 275,732 SEQ ID NO: 3 478,463 SEQ ID NO: 4522,837 SEQ ID NO: 5 443,830 SEQ ID NO: 6 416,905 SEQ ID NO: 7 369,337

These results show that all of the capture oligomers sufficientlypurified HAV RNA from samples to be amplified and detected to indicatethe presence of HAV in the samples.

Example 4 Detection of HAV in Plasma Samples

This example uses an assay that detected HAV nucleic acid inHAV-positive plasma samples. To prepare samples, a commerciallyavailable stock of HAV in human plasma was diluted into HAV-negativeplasma to obtain samples with a titer of 25, 30, 100, 300 and 500copies/ml; a negative control was plasma with no HAV. For each assay,performed using 20 replicate samples per assay, 0.5 ml samples weremixed with 0.4 ml of target capture reagent containing capture oligomersof SEQ ID NO:4 (6.5 pmol/reaction) and SEQ ID NO:5 (1.3 pmol/reaction)and the target capture step was performed substantially as described inExample 3, except that the 60.deg.C. incubation was for 20 min. For eachassay, the washed magnetic particles with the attached hybridizationcomplexes that included capture oligomers of SEQ ID NOS:4 and 5 bound toHAV RNA were then used in the amplification reactions that contained75.micro.l of amplification reagent containing amplification oligomersof SEQ ID NO:36 (13 pmol/reaction) and SEQ ID NO:96 (20 pmol/reaction).As described above, the mixture was covered with an oil layer, incubated10 min at 60.deg.C., the enzyme reagent (25.micro.l) was added, and themixture was incubated for 60 min at 41.5.deg.C. to allow amplificationof the HAV target sequence. The amplified sequences were detected byusing 2-methyl-AE-labeled detection oligomers of SEQ ID NOS:121 and 122(0.007-0.13 pmol/reaction of each in 25.micro.l volume of probe reagent)which were incubated for 15 min at 60.deg.C. for hybridization of theprobes to the amplified HAV sequences, and then 250.micro.l of selectionreagent was added and the mixture was incubated 10 min at 60.deg.C. tohydrolyze AE in unbound probes, and detection was performed as describedabove using the Detect Reagents I and II to produce the chemiluminescentsignal (RLU) measured in a luminometer (LEADER.sup.™ HC Plus, Gen-ProbeInc., San Diego, Calif.). The results showed that the assay has asensitivity of about 80% to 100% for samples containing 25 copies of HAVper ml, about 90% to 100% for samples containing 30 copies of HAV perml, about 98% to 100% for samples containing 100 copies of HAV per ml,and 100% for samples containing 300 and 500 copies of HAV per ml. Nopositive results were detected for the negative control (samplescontaining 0 copies of HAV). These results show that the assay detectsHAV in clinical samples with a sensitivity of about 25 copies of HAV perml of sample.

Example 5 Detection of HAV RNA and Another Viral Target in the SameSample

This example describes an assay that includes the steps of targetcapture, amplification and detection substantially as described inExamples 1 to 4 to detect HAV and similar steps using additionaloligomers to capture, amplify and detect another target, humanparvovirus B19, in the same sample. To detect HAV, the capture oligomersare of SEQ ID NOS:4 and 5, the amplification oligomers are of SEQ IDNOS:36 and 96, and the detection probes are of SEQ ID NOS:121 and 122,substantially as described in Example 4. To detect parvovirus B19nucleic acid, the capture probe is of SEQ ID NO:169, the amplificationoligomers are of SEQ ID NOS:170 and 171, and the detection probes are ofSEQ ID NO:173 labeled between residues 5 and 6 and SEQ ID NO:174 labeledbetween residues 9 and 10. The assay to detect parvovirus B19 is similarto a previously described assay (U.S. Pat. Pub. No. US-2003-0124578-A1).Samples of 0.5 ml normal human plasma are prepared to contain knownamounts of HAV and parvovirus B19, and then mixed with 0.4 ml of targetcapture reagent containing all of the capture oligomers described above.Target capture is performed substantially as described in Example 2. Thepurified HAV and parvovirus B19 targets are amplified in the sameamplification reaction mixture, substantially as described in Examples 3and 4, but using the combination of HAV-specific and parvovirus-specificamplification oligomers described above. Following amplification of theHAV target sequence and the parvovirus B19 target sequence, thedetection step uses the HAV-specific probes and parvovirus-specificprobes as described above, but where the probes for the different targetviruses are each labeled with a different acridinium ester compound toallow detection of different signals in the detection reaction by usingdifferential kinetics (as described previously, see U.S. Pat. No.5,658,737). In these assays, both the HAV and the parvovirus B19 nucleicacid are detected in the samples that contain both targets. Thesensitivity of the assay for detection of HAV is about 25 copies/ml,i.e., positive signals are detected for 80% to 100% of the samplescontaining 25, 30, 100, 300 and 500 copies/ml of HAV. The sensitivity ofthe assay for detection of parvovirus B19 in the sample is as low as 150international units/ml (IU/ml), i.e., positive signals are detected for20% to 40% of samples containing 150 IU/ml. The assay reliably detects400 or more parvovirus B19 IU/ml, i.e., 70% to 100% positive detectionfor samples containing 400, 600, 800, 1600 and 3000 IU/ml. These resultsshow that HAV nucleic acid is specifically detected when the sampleincludes HAV and another virus, human parvovirus B19, which is alsospecifically detected.

We claim:
 1. A combination of at least two oligomers for amplifying aHAV target region comprising: for a first HAV target region, an oligomerof about 23 to 26 nt contained in the sequence of SEQ ID NO:138 thatincludes at least the sequence of SEQ ID NO:139 or SEQ ID NO:140, or anoligomer in a size range of about 19 to 25 nt contained in the sequenceof SEQ ID NO: 141 and that contains at least one sequence of SEQ IDNOS:142 to 146, or a promoter primer oligomer in a size range of about50 to 53 nt that includes a HAV target-specific portion of any one ofSEQ ID NOS:21 to 27; for a second HAV target region, an oligomer ofabout 21 to 27 nt contained in the sequence of SEQ ID NO:60 or containedin the sequence of SEQ ID NO:86 that includes at least the sequence ofSEQ ID NO:156, or a promoter primer oligomer in a size range of about 48to 54 nt that includes a HAV target-specific portion of any one of SEQID NOS:29 to 32; for a third HAV target region, an oligomer of about 24to 30 nt contained in the sequence of SEQ ID NO:147 that includes atleast the sequence of SEQ ID NO:148, or is contained in the sequence ofSEQ ID NO:157 and that includes at least the sequence of SEQ ID NO:158,or a promoter primer oligomer that includes a HAV target-specificportion of SEQ ID NO:31 or SEQ ID NO:32; for a fourth HAV target region,an oligomer of about 18 to 27 nt contained in the sequence of SEQ IDNO:93 or SEQ ID NO:95 and that contains at least the sequence of SEQ IDNO:97, SEQ ID NO:159, or SEQ ID NO:160, or a promoter primer oligomerthat includes a HAV target-specific portion of SEQ ID NO:33; for a fifthHAV target region, an oligomer of about 19 to 31 nt contained in thesequence of SEQ ID NO:149 and that includes at least the sequence of SEQID NO:150, or a promoter primer oligomers in a size range of about 51 to56 nt that includes a HAV target-specific portion of any one of SEQ IDNOS:34 to 40; for a sixth HAV target region, an oligomer of about 24 to28 nt contained in the sequence of SEQ ID NO:161 and that include atleast the sequence of SEQ ID NO:162, or a promoter primer oligomer thatincludes a HAV target-specific portion of SEQ ID NO:41 or SEQ ID NO:42;for a seventh HAV target region, an oligomer of about 20 to 30 ntcontained in the sequence of SEQ ID NO: 151 and that includes at leastany one of the sequences of SEQ ID NO:152 to SEQ ID NO:155, or iscontained in SEQ ID NO:163 and includes at least the sequence of SEQ IDNO:164, or is contained in SEQ ID NO:165 and includes at least any oneof the sequences of SEQ ID NOS:166 to 168, or a promoter primer oligomerin a size range of about 51 to 56 nt and that includes a HAVtarget-specific portion of any one of SEQ ID NOS:43 to 49; and a secondamplification oligomer.
 2. The combination of at least two oligomers ofclaim 1 for the first HAV target region selected from the groupconsisting of: SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,SEQ ID NO:57, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83,SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:143, SEQ ID NO:144, and SEQ IDNO:145.
 3. The combination of at least two oligomers of claim 1 for thesecond HAV target region selected from the group consisting of: SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:86, SEQ ID NO:87, SEQ IDNO:88, and SEQ ID NO:156.
 4. The combination of at least two oligomersof claim 1 for the third HAV target region selected from the groupconsisting of: SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:61, SEQ ID NO:62,SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:148.
 5. Thecombination of at least two oligomers of claim 1 for the fourth HAVtarget region selected from the group consisting of: SEQ ID NO:33, SEQID NO:63, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ IDNO:96, and SEQ ID NO:97.
 6. The combination of at least two oligomers ofclaim 1 for the fifth HAV target region selected from the groupconsisting of: SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:64, SEQ ID NO:65,SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70,SEQ ID NO:97, SEQ ID NO:149, and SEQ ID NO:150.
 7. The combination of atleast two oligomers of claim 1 for the sixth HAV target region selectedfrom the group consisting of: SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:161,and SEQ ID NO:162.
 8. The combination of at least two oligomers of claim1 for the seventh HAV target region selected from the group consistingof: SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:73, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ IDNO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:104, SEQ ID NO:106, SEQID NO:107, SEQ ID NO:108, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:155,SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ IDNO:167, and SEQ ID NO:168.
 9. The combination of at least two oligomersof claim 1, further comprising at least one capture probe oligomerselected from the group consisting of SEQ ID NOS:1 to 7 and SEQ IDNOS:8-14 linked to a moiety that allows it to bind a solid support. 10.The combination of at least two oligomers of claim 1, further comprisingat least one detection probe oligomer selected from the group consistingof SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ IDNO:117, SEQ ID NO:119, SEQ ID NO:121 to SEQ ID NO:124, and SEQ ID NO:126to SEQ ID NO:130.
 11. The combination of at least two oligomersaccording to claim 1, wherein said combination is packaged in a kit. 12.A method of detecting the presence of HAV in a sample comprising thesteps of: purifying HAV nucleic acid from other components in a samplecontaining HAV; amplifying a HAV target sequence in the purified HAVnucleic acid, or a cDNA made therefrom, by using an in vitroamplification reaction that includes at least two amplificationoligomers specific for a selected HAV target region, which include: fora first HAV target region, an oligomer of about 23 to 26 nt contained inthe sequence of SEQ ID NO:138 and that includes at least the sequence ofSEQ ID NO:139 or SEQ ID NO:140, or an oligomer in a size range of about19 to 25 nt contained in the sequence of SEQ ID NO: 141 and thatcontains at least one sequence of SEQ ID NOS:142 to 146, or a promoterprimer oligomer in a size range of about 50 to 53 nt that includes a HAVtarget-specific portion of any one of SEQ ID NOS:21 to 27; for a secondHAV target region, an oligomer of about 21 to 27 nt contained in thesequence of SEQ ID NO:60 or contained in the sequence of SEQ ID NO:86and that includes at least the sequence of SEQ ID NO:156, or a promoterprimer oligomer in a size range of about 48 to 54 nt that includes a HAVtarget-specific portion of any one of SEQ ID NOS:29 to 32; for a thirdHAV target region, an oligomer of about 24 to 30 nt contained in thesequence of SEQ ID NO:147 and that includes at least the sequence of SEQID NO:148, or contained in the sequence of SEQ ID NO:157 and thatincludes at least the sequence of SEQ ID NO:158, or a promoter primeroligomer that includes a HAV target-specific portion of SEQ ID NO:31 orSEQ ID NO:32; for a fourth HAV target region, an oligomer of about 18 to27 nt contained in the sequence of SEQ ID NO:93 or SEQ ID NO:95 and thatcontains at least the sequence of SEQ ID NO:97, SEQ ID NO:159, or SEQ IDNO:160, or a promoter primer oligomer that includes a HAVtarget-specific portion of SEQ ID NO:33; for a fifth HAV target region,an oligomer of about 19 to 31 nt contained in the sequence of SEQ IDNO:149 and that includes at least the sequence of SEQ ID NO:150, or apromoter primer oligomer in a size range of about 51 to 56 nt thatincludes a HAV target-specific portion of any one of SEQ ID NOS:34 to40; for a sixth HAV target region, an oligomer of about 24 to 28 ntcontained in the sequence of SEQ ID NO:161 and that includes at leastthe sequence of SEQ ID NO:162, or a promoter primer oligomer thatincludes a HAV target-specific portion of SEQ ID NO:41 or SEQ ID NO:42;for a seventh HAV target region, an oligomer of about 20 to 30 ntcontained in the sequence of SEQ ID NO: 151 and that includes at leastany one of the sequences of SEQ ID NO:152 to SEQ ID NO:155, or iscontained in SEQ ID NO:163 and includes at least the sequence of SEQ IDNO:164, or is contained in SEQ ID NO:165 and includes at least any oneof the sequences of SEQ ID NOS:166 to 168, or a promoter primer oligomerin a size range of about 51 to 56 nt and that includes a HAVtarget-specific portion of any one of SEQ ID NOS:43 to 49; and a secondamplification oligomer; to produce an amplified product of the selectedHAV target region; and detecting the amplified product.
 13. The methodof claim 12, wherein the purifying step contacts the sample with atleast one capture probe oligomer selected from the group consisting ofSEQ ID NOS:1 to 7 and SEQ ID NOS:8-14 linked to a moiety that allows itto bind a solid support, wherein a target hybridization sequence of saidat least one capture probe oligomer hybridizes specifically to asequence in HAV RNA to form a hybridization complex with the HAV RNA,and can be used with a solid support to separate the HAV RNA from othersample components.
 14. The method of claim 12, wherein the amplifyingstep amplifies a sequence in the first HAV target region by using atleast two oligomers specific for the first HAV target region selectedfrom the group consisting of: SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82,SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:143, SEQ ID NO:144,and SEQ ID NO:145; and wherein the detecting step uses at least onedetection probe that hybridizes specifically to the amplified product ofthe first HAV target region.
 15. The method of claim 12, wherein theamplifying step amplifies a sequence in the second HAV target region byusing at least two oligomers specific for the second HAV target regionselected from the group consisting of: SEQ ID NO:28, SEQ ID NO:29, SEQID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:156; andwherein the detecting step uses at least one detection probe thathybridizes specifically to the amplified product of the second HAVtarget region.
 16. The method of claim 12, wherein the amplifying stepamplifies a sequence in the third HAV target region by using at leasttwo oligomers specific for the third HAV target region selected from thegroup consisting of: SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:148; andwherein the detecting step uses at least one detection probe thathybridizes specifically to the amplified product of the third HAV targetregion.
 17. The method of claim 12, wherein the amplifying stepamplifies a sequence in the fourth HAV target region by using at leasttwo oligomers specific for the fourth HAV target region selected fromthe group consisting of: SEQ ID NO:33, SEQ ID NO:63, SEQ ID NO:92, SEQID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, and SEQ ID NO:97;and wherein the detecting step uses at least one detection probe thathybridizes specifically to the amplified product of the fourth HAVtarget region.
 18. The method of claim 12, wherein the amplifying stepamplifies a sequence in the fifth HAV target region by using at leasttwo oligomers specific for the fifth HAV target region selected from thegroup consisting of: SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:64, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:97, SEQ ID NO:149, and SEQ ID NO:150; and wherein thedetecting step uses at least one detection probe that hybridizesspecifically to the amplified product of the fifth HAV target region.19. The method of claim 12, wherein the amplifying step amplifies asequence in the sixth HAV target region by using at least two oligomersspecific for the sixth HAV target region selected from the groupconsisting of: SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:71, SEQ ID NO:72,SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:161, and SEQ IDNO:162; and wherein the detecting step uses at least one detection probethat hybridizes specifically to the amplified product of the sixth HAVtarget region; or wherein the amplifying step amplifies a sequence inthe seventh HAV target region by using at least two oligomers specificfor the seventh HAV target region selected from the group consisting of:SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75,SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:102,SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:104, SEQ ID NO:106, SEQ IDNO:107, SEQ ID NO:108, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:155, SEQID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167,and SEQ ID NO:168; and wherein the detecting step uses at least onedetection probe that hybridizes specifically to the amplified product ofthe seventh HAV target region.
 20. The method of claim 12, wherein saiddetecting step uses a nucleic acid detection oligomer, wherein saidnucleic acid detection oligomer comprises a target hybridizing sequencethat specifically hybridizes with at least one amplified productgenerated in said amplifying step; optionally wherein said nucleic aciddetection oligomer comprises a target hybridizing region nucleic acidsequence consisting essentially of a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121 to SEQ ID NO:124,and SEQ ID NO:126 to SEQ ID NO:130; optionally wherein said nucleic aciddetection oligomer further comprises a 2′-O-methyl linkages, achemiluminescent label, an acridinium ester label, a synthetic linker orcombinations thereof.